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Characterization of the P 2Y ‐purinoceptor involved in the ATP‐induced rise in cytosolic Ca 2+ concentration in rat ileal myocytes
Author(s) -
Pacaud Pierre,
Feolde Erick,
Frelin Christian,
Loirand Gervaise
Publication year - 1996
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1996.tb15665.x
Subject(s) - ppads , carbachol , agonist , inositol phosphate , phospholipase c , p2y receptor , stimulation , inositol , endocrinology , medicine , cytosol , biophysics , myocyte , chemistry , biology , biochemistry , receptor , enzyme
1 The P 2 ‐purinoceptor subtype and the intracellular signalling mechanism(s) involved in the rise in the free cytosolic Ca 2+ concentration ([Ca 2+ ] i ) induced by ATP and analogues were analyzed in myocytes isolated from the longitudinal muscle layer of rat ileum by means of molecular and physiological techniques. 2 The P 2 ‐purinoceptor expressed by ileal smooth muscle cells shared 100% amino acid identity with the rat P 2Y1 ‐receptor. 3 Short applications of the purinoceptor agonists induced a transient rise in [Ca 2+ ] i in an all‐or‐nothing manner. The rank order of potency of the analogues of ATP and ADP, determined by measuring the percentage of responding cells was 2‐methylthioATP = 2‐chloro‐ATP > ADP > ATP, with concentrations giving [Ca 2+ ] i response in 50% of cells ranging between 3 nM and 0.6 μ m . The concentration‐response curves to ADP and ATP were shifted to the right by 10 μ m pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS). 4 Although the rise in [Ca 2+ ] i induced by stimulation of the ileal P 2Y ‐purinoceptor was inhibited by heparin (5 mg ml −1 ), we were not able to detect stimulation of phospholipase C under conditions (37°C) where muscarinic cholinoceptor activation markedly increased inositol phosphate (InsP) accumulation. However, the carbachol (CCh)‐induced increase in InsP accumulation was suppressed when the agonist was applied at 20°C while a CCh‐induced [Ca 2+ ] i rise similar to that obtained in response to the P 2 ‐purinoceptor agonist was still observed. 5 Our results indicate that the rat ileal myocytes express a PPADS‐sensitive P 2 ‐purinoceptor similar to the P 2Y1 ‐receptor subtype. Although there is no detectable increase in InsP production, stimulation of these receptors leads to a rise in [Ca 2+ ] i by activation of the inositol 1,4,5‐trisphosphate receptor‐channel of the intracellular Ca 2+ store, indicating that they couple to phospholipase C.